Primary biliary cirrhosis is a severe, progressive, autoimmune liver disease of unknown etiology characterized by the presence of antibodies to mitochondria. These antibodies are directed against a complex of proteins of 70, 45 and 39 kd, found in the mitochondria of all human tissues and in the mitochondria of many other species. More than 95% of PBC patients have antibodies to at least one of these proteins, most commonly to the 70 kd protein. An understanding of the clinical relevance of the antimitochondrial response found in PBC requires that these proteins be characterized at the molecular level. We have cloned and sequenced a 1.4 kb cDNA from a rat liver library that encodes part of the 70 kd mitochondrial antigen. The 1.4 kb cDNA expresses a fragment of the 70 kd antigen as a fused polypeptide that is capable of absorbing all anti 70 kd antibodies from patient serum. We have used this probe to begin cloning the human cDNA equivalent. Finally, we have shown that this fused polypeptide, when injected into mice, elicits a-specific antimitochondrial response. Using these tools, we will determine the complete sequence of the 70 kd mitochondrial gene in both the rat and human. We will identify regions in the human sequence that are the binding sites for autoantibodies found in PBC patients. We will synthesize peptides corresponding to these sequences and use them to assay titer and class of patient antibody and determine any relationship to prognosis. We will similarly identify regions in the human sequence that stimulate peripheral blood T lymphocytes of PBC patients and synthesize peptides corresponding to these regions. Some patients with PBC also have antibodies to a 45 and 39 kd mitochondrial autoantigen. We will clone these genes and determine relatedness of their sequences to that of the full length 70 kd gene. Finally, we will begin a pilot program to immunize mice with the purified fused polypeptide and monitor T cell reactivity, antibody responses and liver histology. The identification of reactive epitopes for B and T cells will allow the generation